Ptgdr-1 and/or ptgdr-2 antagonists for preventing and/or treating systemic lupus erythematosus

ABSTRACT

The present invention concerns a PTGDR-1 antagonist, a PTGDR-2 antagonist, a dual PTGDR-1/PTGDR-1 antagonist, or a combination of PTGDR-1 antagonist and PTGDR-2 antagonist, and pharmaceutical compositions containing them, for use for preventing and/or treating SLE.

The present invention concerns a PTGDR-1 antagonist, a PTGDR-2antagonist, a dual PTGDR-1/PTGDR-1 antagonist, or a combination ofPTGDR-1 antagonist and PTGDR-2 antagonist, and pharmaceuticalcompositions containing them, for use for preventing and/or treatingSLE.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmunedisease which can affect various organs such as joints and skin and belethal when kidney involvement (lupus nephritis, LN) is not controlled.If considered as a B cell disease, both innate and adaptive immunesystems are dysregulated during SLE and synergize to amplify theproduction of the main lupus pathogenic factors which are autoantibodiesmostly directed against nuclear antigens (ANA) such as double strandedDNA (dsDNA). Once aggregated to their antigens and complement factors,these autoantibodies will form circulating immune complexes mediating achronic inflammation when deposited in the targeted organ. Flares of thedisease are usually controlled by strong immunosuppressive treatmentsand high dose of corticosteroids. Recent clinical trials aimed todecrease autoantibody production in subjects with SLE by directlytargeting the B cell compartment but failed to demonstrate sufficientefficacy. Developing new therapeutic strategies to prevent flares tooccur represents a big challenge for the biomedical community.

Basophils are one of the less represented circulating leukocytes and arewell known for their involvement in allergic and parasitic diseases.During the past decade, basophils were shown having powerful immuneregulatory functions despite their weak representation). It waspreviously shown that basophils were able to support plasma cellsurvival and antibody production in vivo while expressing some B cellactivating factors such as BAFF (B cell activating factor), CD40L (CD40ligand or CD154), interleukin (IL)-4 and IL-6 ((Voehringer, D., Nat.Rev. Immunol. 13, 362-375 (2013); Charles, N. et al., Nat. Med. 16,701-707 (2010)). This immunomodulatory role is associated with theirability to accumulate in secondary lymphoid organs (SLOs) where they canhelp T and B cells in differentiation and maturation (Charles, N. etal., Nat. Med. 16, 701-707 (2010); Leyva-Castillo, J. M., et al., NatCommun 4, 2847 (2013); Otsuka, A., et al., Nat Commun 4, 1739 (2013)).Mechanisms leading to SLOs basophil accumulation are poorly understood.

C—X—C motif Ligand 12 (CXCL12) is a chemokine secreted mostly by stromalcells from bone marrow, peritoneal cavity, SLOs, and kidneys. CXCL12acts as a homeostatic chemokine by regulating mesenchymal stem cells, Bcells and neutrophils physiological distribution via its specificinteraction with C—X—C motif Receptor 4 (CXCR4). CXCL12 overexpressionoccurs in inflamed tissues and is pathogenic in cancer and lupusnephritis among other diseases (Wang, A., et al., J. Immunol. 182,4448-4458 (2009); Balabanian, K., et al., J. Immunol. 170, 3392-3400(2003); Hummel, S., Van Aken, H. & Zarbock, A. Curr. Opin. Hematol. 21,29-36 (2014)).

Prostaglandin D₂ (PGD₂) is produced from arachidonic acid bycyclooxygenases and tissue-specific PGD₂ synthases (PGDS). PGD₂contributes to various homeostatic functions and is involved in theonset and resolution of inflammation through its interactions with thetwo known PGD₂ receptors (PTGDRs): PTGDR-1 (or DP, D prostanoidreceptor) and PTGDR-2 (or DP-2, also known as chemoattractantreceptor-homologous molecule expressed on T helper type 2 (T_(H)2)cells, CRTH2). Basophils express the highest level of the ubiquitousPTGDR-1 among peripheral blood leukocytes. PTGDR-2 expression is morerestricted and mediates activation and chemotaxis of basophils,eosinophils and T_(H)2 CD4⁺ T cells. The effects of these two receptorscan be competitive or cooperative. PGD₂ has been involved in allergicand pulmonary diseases, ulcerative colitis, and renal fibrosis.Lipocalin-type-PGDS (L-PGDS) was recently found to be expressed de novoin inflamed kidneys (Nagata, N., et al., FEBS J 276, 7146-7158 (2009)and in the urine of active LN patients (Suzuki, M., et al., Pediatr.Res. 65, 530-536 (2009); Somparn, P., et al., J. Proteomics 75,3240-3247 (2012)).

However, the PGD₂/PTGDRs axis has not been characterized in SLE.

In this respect, the international patent application WO 2009/085177 hasdisclosed dual PTGDR-1/PTGDR-2 antagonists and suggested that theseantagonists could be useful in the prevention and/or treatment of a listof diseases including SLE. However the only therapeutic effectconcretely evaluated in this document is against asthma. The proposedprevention and/or treatment of SLE is not substantiated.

The inventors recently showed that basophils were involved in thedevelopment of LN both in a spontaneous murine SLE model (Lyn^(−/−)mice) and in a small cohort of 42 patients by accumulating in SLOs wherethey support autoreactive T and B cells through an IgE and IL-4dependent pathway (Charles, N. et al., Nat. Med. 16, 701-707 (2010)).Since the production of potent basophil activators or chemo-attractantsis known to be dysregulated during lupus pathogenesis (Pellefigues, C. &Charles, N., Curr. Opin. Immunol. 25, 704-711 (2013)), mechanismsunderlying basophil recruitment to SLOs during SLE were explored.

The inventors have now identified two new pathways by which basophilsget activated during flares of the disease.

Tissue chronic inflammation is leading to the secretion of basophilactivating factors which systemic concentrations are dysregulated duringlupus. In a new larger cohort of individuals with SLE, the inventorsconfirmed that basopenia was a characteristic of SLE patients and wascorrelated with disease activity. Moreover, as compared to other renaldiseases, basopenia was specific of lupus nephritis. This basopenia wasassociated with a specific expression pattern on basophils of some knownand new activation markers. Indeed, CXCR4 expression at the proteinlevel and on the surface of basophils was markedly increased in activepatients and associated with basopenia.

The inventors found that disease activity and basopenia were tightlylinked together and to PGD₂/PTGDRs and CXCL12/CXCR4 axes.

Beyond their altered expression pattern on active lupus subjectbasophils, CXCR4 and CD164 led to a dramatically increased sensitivityof SLE basophils to CXCL12-mediated migration ex vivo. In non-sterileperitonitis patients, the inventors demonstrated that migrated basophilswere dramatically overexpressing CXCR4 as compared to their“non-migrated” blood basophil counterparts, strongly suggesting thathuman CXCR4⁺ basophil extravasation and migration could be induced invivo by CXCL12, leading to a peripheral basopenia. In mice, CXCL12induced a redistribution of CXCR4⁺ basophils at the injection site andin the corresponding draining lymph nodes, demonstrating basophilmigration capability to CXCL12 in vivo. These migration abilities, bothin human and mice, were enabled in vivo and ex vivo by the PGD₂/PTGDRsaxis.

Furthermore, the inventors demonstrated that PGD₂ synthesis wasincreased in SLE subjects and associated with basopenia. ThisPGD₂-mediated CXCR4-dependent basophil migration was, at leastpartially, due to an autocrine effect of PGD₂ upon its synthesis bybasophils themselves ex vivo, resulting in an externalization of CXCR4.In particular, both in subjects with SLE and in Lyn^(−/−) mice, theinventors found that PGD₂ (the PGD₂/PTGDRs axis) was enhancingCXCR4-dependent basophil recruitment to SLOs during lupus, explainingthe flare of the disease observed with repeated PGD₂ injections inlupus-prone mice. These data underline the CXCR4-mediated pathogeniceffect of PGD₂ during lupus pathogenesis, identifying both axes asputative therapeutic targets.

The inventors thus concluded that altering these axes in SLE patientsusing PTGDRs specific antagonists should break the basophil-dependentautoantibody production and kidney inflammation as showed in theLyn^(−/−) lupus-like mouse model, and should limit SLE flares and longterm organ damages by preventing basophil homing to SLOs.

The inventors demonstrated that antagonizing PTGDRs in vivo in alupus-like mouse model prevented the CXCR4-dependent basophilrecruitment in SLOs without impacting other cell type proportions butthe short lived plasma cells. This treatment achieved to dampenshort-lived plasma cell number, renal inflammation, and autoantibodiestiters in only 10 days.

Definitions

The terms “lupus” or “systemic lupus erythematosus” or “SLE” are used intheir customary meaning herein and include an autoimmune disordercharacterized by the presence of autoantibodies, rash, oral ulcers,serositis, neurological disorders, low blood cell counts, joint pain andswelling. Tests used to diagnose include antibody tests (e.g.,Antinuclear antibody (ANA) panel, Anti-double strand DNA (dsDNA),Antiphospholipid antibodies, Anti-Smith antibodies); CBC to show lowwhite blood cells, hemoglobin, or platelets; chest x-ray showingpleuritis or pericarditis; kidney biopsy; urinalysis to show blood,casts, or protein in the urine.

By “lupus nephritis” is meant a disorder characterized by aninflammation of the kidney caused by systemic lupus erythematosus (SLE).Lupus nephritis is characterized by IgM-, IgG-, and IgA-containingimmune complexes deposited in the glomeruli. These immune complexes areformed by autoantibodies with specificity to nuclear components(antinuclear antibodies (ANA)) or to nucleic acids (such asdouble-stranded DNA (dsDNA)).

Accordingly, as used herein, prevention and/or treatment of systemiclupus erythematosus or “SLE” encompasses prevention and/or treatment oflupus nephritis.

In the context of the invention, the term “treating” or “treatment”,refers to a therapeutic use (i.e. on a subject having a given disease)and means reversing, alleviating, inhibiting the progress of one or moresymptoms of such disorder or condition. Therefore, treatment does notonly refer to a treatment that leads to a complete cure of the disease,but also to treatments that slow down the progression of the diseaseand/or prolong the survival of the subject.

By “preventing” is meant a prophylactic use (i.e. on a subjectsusceptible of developing a given disease).

“Prostaglandin D2” or “PDG₂” is9α,15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic acid (IUPAC name) (CASnumber: 41598-07-6).

“PTGDR-1” or “DP” or “D prostanoid receptor” is a receptor forprostaglandin D2 and its activity is mainly mediated by G(s) proteinsthat stimulate adenylate cyclase, resulting in an elevation ofintracellular cAMP. An exemplary sequence of human PTGDR-1 protein isavailable in the UniProt database, under accession number Q13258 (inparticular Q13258-1:isoform 1, Entry version 133 of 7 Jan. 2015). Acomplete mRNA sequence encoding PTGDR-1 is available in Genbank underaccession number EF577397.1 (release: 122; issue date: 21 Nov. 2014).

“PTGDR-2” or “chemoattractant receptor-homologous molecule expressed onT helper type 2 (TH2) cells” or “CRTH2” or “DP2” is a G-protein-coupledreceptor for prostaglandin D2 that is preferentially expressed in CD4⁺effector T helper 2 (Th2) cells. An exemplary sequence of human PTGDR-2protein is available in the UniProt database, under accession numberQ9Y5Y4 (Entry version 120 of 7 Jan. 2015). A complete mRNA sequenceencoding PTGDR-2 is available in Genbank under accession numberAY507142.1 (release: 122; issue date: 21 Nov. 2014).

As used herein, the term “antagonist” refers to any molecule thatpartially or fully blocks, inhibits, reduces or neutralizes expressionand/or biological activity of a target and/or signalling pathway.

“PTGDR-1 antagonist” refers to a molecule that partially or fullyblocks, inhibits, neutralizes, or interferes with the expression and/orbiological activities of a PTGDR-1 protein. This includes, but is notlimited to, blocking, inhibiting, reducing, or interfering withPDG₂/PTGDR-1 interactions. PTGDR-1 antagonists include for instance aPDG₂ neutralising antibody, i.e. an antibody which binds to PDG₂ andprevents PDG₂ binding to PTGDR-1.

“PTGDR-2 antagonist” refers to a molecule that partially or fullyblocks, inhibits, neutralizes, or interferes with the expression and/orbiological activities of a PTGDR-2 protein. This includes, but is notlimited to, blocking, inhibiting, reducing, or interfering withPDG₂/PTGDR-2 interactions. PTGDR-2 antagonists include for instance aPDG₂ neutralising antibody, i.e. an antibody which binds to PDG₂ andprevents PDG₂ binding to PTGDR-2.

Suitable antagonist molecules specifically include, but are not limitedto biological molecules such as a protein, polypeptide, peptide,antibody, antibody fragment, aptamers, antisense, interfering RNAs, ornon-biological large or small molecules (less than 10 kDa), inparticular small organic molecules.

The antagonist may be an inhibitor of PTGDR-1 or PTGDR-2 geneexpression. Inhibitors of gene expression include antisenseoligonucleotides and interfering RNA (iRNA).

The term “iRNA” include double-stranded RNA, single-stranded RNA,isolated RNA (partially purified RNA, essentially pure RNA, syntheticRNA, recombinantly produced RNA), as well as altered RNA that differsfrom naturally occurring RNA by the addition, deletion, substitutionand/or alteration of one or more nucleotides. Such alterations caninclude addition of non-nucleotide material, such as to the end(s) ofthe RNA or internally (at one or more nucleotides of the RNA).Nucleotides in the iRNA molecules can also comprise non-standardnucleotides, including non-naturally occurring nucleotides ordeoxyribonucleotides. Collectively, all such altered iRNA compounds arereferred to as analogs or analogs of naturally-occurring RNA. A iRNAneeds only be sufficiently similar to natural RNA that it has theability to mediate RNA interference. As used herein the phrase “mediateRNA Interference” refers to and indicates the ability to distinguishwhich mRNA are to be affected by the RNA interference machinery orprocess. RNA that mediates RNA interference interacts with the RNAinterference machinery such that it directs the machinery to degradeparticular mRNAs or to otherwise reduce the expression of the targetprotein. In one embodiment, iRNA molecules direct cleavage of specificmRNA to which their sequence corresponds. It is not necessary that therebe perfect correspondence of the sequences, but the correspondence mustbe sufficient to enable the iRNA to direct RNA interference inhibitionby cleavage or lack of expression of the target mRNA. The iRNA moleculesmay comprise an RNA portion and some additional portion, for example adeoxyribonucleotide portion. The total number of nucleotides in the RNAmolecule is suitably less than 49 in order to be effective mediators ofRNA interference. In preferred RNA molecules, the number of nucleotidesis 16 to 29, more preferably 18 to 23, and most preferably 21-23.

As indicated above, the term iRNA includes but is not limited to siRNAs,shRNAs, miRNAs, dsRNAs, and other RNA species that can be cleaved invivo to form siRNAs.

A “short interfering RNA” or “siRNA” comprises a RNA duplex(double-stranded region) and can further comprises one or twosingle-stranded overhangs, 3′ or 5′ overhangs.

A “short hairpin RNA (shRNA)” refers to a segment of RNA that iscomplementary to a portion of a target gene (complementary to one ormore transcripts of a target gene), and has a stem-loop (hairpin)structure.

“MicroRNAs” or “miRNAs” are endogenously encoded RNAs that are about22-nucleotide-long, that post-transcriptionally regulate target genesand are generally expressed in a highly tissue-specific ordevelopmental-stage-specific fashion. One can design and expressartificial miRNAs based on the features of existing miRNA genes. ThemiR-30 (microRNA 30) architecture can be used to express miRNAs (orsiRNAs) from RNA polymerase II promoter-based expression plasmids (Zenget al, 2005, Methods enzymol. 392:371-380). In some instances theprecursor miRNA molecules may include more than one stem-loop structure.The multiple stem-loop structures may be linked to one another through alinker, such as, for example, a nucleic acid linker, a miRNA flankingsequence, other molecules, or some combination thereof.

Anti-sense oligonucleotides are non-enzymatic nucleic acid moleculesthat bind to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA(protein nucleic acid; Egholm et al, 1993 Nature 365, 566) interactionsand alter the activity of the target RNA. Typically, anti-sensemolecules are complementary to a target sequence along a contiguoussequence of the antisense molecule. In addition, anti-sense DNA can beused to target RNA by means of DNA-RNA interactions, thereby activatingRNase H, which digests the target RNA in the duplex.

The term “antibody” refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site which immunospecificallybinds an antigen. As such, the term antibody encompasses not only wholeantibody molecules, but also antibody fragments as well as variants(including derivatives) of antibodies and antibody fragments. Inparticular, the antibody according to the invention may correspond to apolyclonal antibody, a monoclonal antibody (e.g. a chimeric, humanizedor human antibody), a fragment of a polyclonal or monoclonal antibody ora diabody.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fv, Fab, F(ab′)₂, Fab′,Fd, dAb, dsFv, scFv, sc(Fv)₂, CDRs, diabodies and multi-specificantibodies formed from antibodies fragments.

“Aptamers” are a class of molecule that represents an alternative toantibodies in term of molecular recognition. Aptamers areoligonucleotide or oligopeptide sequences with the capacity to recognizevirtually any class of target molecules with high affinity andspecificity. Such ligands may be isolated through Systematic Evolutionof Ligands by EXponential enrichment (SELEX) of a random sequencelibrary, as described in Tuerk C. and Gold L., Science, 1990,249(4968):505-10. The random sequence library is obtainable bycombinatorial chemical synthesis of DNA. In this library, each member isa linear oligomer, eventually chemically modified, of a unique sequence.Possible modifications, uses and advantages of this class of moleculeshave been reviewed in Jayasena S. D., Clin. Chem., 1999, 45(9):1628-50.Peptide aptamers consists of a conformationally constrained antibodyvariable region displayed by a platform protein, such as E. coliThioredoxin A that are selected from combinatorial libraries by twohybrid methods (Colas et al., Nature, 1996, 380, 548-50).

Throughout the instant application, the term “comprising” is to beinterpreted as encompassing all specifically mentioned features as welloptional, additional, unspecified ones. As used herein, the use of theterm “comprising” also discloses the embodiment wherein no featuresother than the specifically mentioned features are present (i.e.“consisting of”).

Furthermore the indefinite article “a” or “an” does not exclude aplurality.

DESCRIPTION OF THE INVENTION

The invention relates to a PTGDR-1 antagonist for use in a method forpreventing and/or treating SLE. The invention also relates to the use ofa PTGDR-1 antagonist for the manufacture of a medicament for preventingand/or treating SLE. The invention further relates to a method forpreventing and/or treating SLE in a patient in need thereof, whereinsaid method comprises administering said patient with a PTGDR-1antagonist.

According to another aspect, the invention relates to a PTGDR-2antagonist for use in a method for preventing and/or treating SLE. Theinvention also relates to the use of a PTGDR-2 antagonist for themanufacture of a medicament for preventing and/or treating SLE. Theinvention further relates to a method for preventing and/or treating SLEin a patient in need thereof, wherein said method comprisesadministering said patient with a PTGDR-2 antagonist.

PTGDR-1 Antagonists

According to an embodiment, said PTGDR-1 antagonist is a small moleculeantagonist.

Numerous PTGDR-1 antagonists have been described in the art.

Said small molecule antagonist is in particular selected from the groupconsisting of compounds of formulae I to VI as disclosed hereafter:

(i) Compounds of formula (I) as described in patent applicationWO03062200:

-   -   and pharmaceutically acceptable salts thereof, wherein    -   n is 0 or 1; m is 1, 2 or 3; R₁ is H, C₁-C₃ alkyl, halogenated        C₁-C₃ alkyl or cyclopropyl; R₂ is 4-chlorophenyl or        2,4,6-trichlorophenyl.    -   Preferably, the compounds of formula I have the        stereoconfiguration shown below (i.e. the chiral center has the        R configuration):

In particular, the compound of formula I or II is laropiprant, i.e.(−)-[4-(4-chlorobenzyl)-7-fluoro-5-(methane-sulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]aceticacid and pharmaceutically acceptable salts thereof:

Laropiprant is approved by the FDA to inhibit the flushing induced byniacin to treat dyslipidemias.

(ii) Compounds disclosed in patent application WO0179169: i.e.2-[(1R)-9-(4-chlorobenzyl)-8-((R)-methyl-sulfinyl)-2,3,4,9-tetrahydro-1H-carbazol-1-yl]acetic acid, or a pharmaceutically acceptable salt thereof, or2-[(1R)-9-(4-chlorobenzyl)-8-((S)-methyl-sulfinyl)-2,3,4,9-tetrahydro-1H-carbazol-1-yl]acetic acid, or a pharmaceutically acceptable salt thereof;

(iii) Compounds disclosed in patent application WO0208186:

and pharmaceutically acceptable salts, hydrates and esters thereof,wherein:

R1, R2 and R3 are each independently selected from the group consistingof: (1) hydrogen, and (2) Rc,

R4 and R5 are each independently selected from the group consisting of:(1) H, (2) F, (3) CN, (4) C1-6alkyl, (5) ORa, and (6) S(O)_(n)C1-6alkyl,wherein each of said alkyl group is optionally substituted with halogen,or

R4 and R5 on the same carbon atom represent an oxo, or

R4 and R5 on the same carbon atom or on adjacent carbon atoms takentogether form a 3- or 4-membered ring containing 0 or 1 heteroatomselected from N, S, or O optionally substituted with one or two groupsselected from F, CF₃ and CH₃;

R6 is selected from the group consisting of: (1) H, (2) C1-6alkyloptionally substituted with one to six groups independently selectedfrom ORa and halogen, and (3) heterocyclyl optionally substituted withone to four halogen; or

R5 and R6 attached on adjacent carbon atoms together form a 3- or4-membered ring containing 0 or 1 heteroatom selected from N, S, or Ooptionally substituted with one or two groups selected from F, CF₃ andCH₃;

X is selected from the group consisting of: C═O, SO₂, and C1-4alkylwherein said alkyl is optionally substituted with one to six halogen;

Ar is aryl or heteroaryl each optionally substituted with one to fourgroups independently selected from Rc;

Q is C1-6alkyl optionally substituted with one to six groupsindependently selected from: (1) halogen, (2) aryl, (3) heteroaryl, (4)OH, (5) OC1-6alkyl, (6) COOH, (7) CONRaRb, (8) C(O)NSO₂R7, (9)tetrazolyl, wherein aryl, heteroaryl and alkyl are each optionallysubstituted with one to six groups independently selected from halogen,CF₃, and COOH; or

Q and R6 together form a 3- or 4-membered ring optionally containing aheteroatom selected from N, S, and O, and optionally substituted withone or two groups independently selected from: (1) halogen, (2) oxo, (3)ORa, (4) COOH, (5) C(O)NHSO₂R7, and (6) tetrazolyl,

R7 is selected from the group consisting of: (1) C1-6alkyl optionallysubstituted with one to six halogen, (2) aryl, and (3) heteroaryl,wherein said aryl and heteroaryl are optionally substituted withhalogen, OC1-5alkyl, C1-5alkyl and wherein said alkyl is optionallysubstituted with one to six halogen;

Ra and Rb are independently selected from hydrogen and C1-6alkyloptionally substituted with one to six halogen;

Rc is (1) halogen, (2) CN, (3) C1-6alkyl optionally substituted with oneto six groups independently selected from halogen, NRaRb, C(O)Ra,C(ORa)RaRb, and ORa, (4) C2-6alkenyl optionally substituted with one tosix groups independently selected from halogen and ORa, (5)heterocyclyl, (6) aryl, (7) heteroaryl, (8) C(O)Ra, (9) C(ORa)RaRb (10)C(O)ORa, (11) CONRaRb, (12) OCONRaRb, (13) S(O)_(n)R7, (14)NRaC(O)OC1-6alkyl, wherein alkyl is optionally substituted with one tosix halogen and (15) S(O)_(n)NRaRb, wherein heterocyclyl, aryl,heteroaryl are optionally substituted with one to four groupsindependently selected from halogen; n is 0, 1 or 2.

(iv) Compounds disclosed in patent application WO03062200:

-   -   and pharmaceutically acceptable salts thereof, wherein    -   n is 0 or 1; m is 1, 2 or 3; R₁ is H, C₁-C₃ alkyl, halogenated        C₁-C₃ alkyl or cyclopropyl; R₂ is 4-chlorophenyl or        2,4,6-trichlorophenyl.    -   In particular, a compound of formula IV is        (+[4-(4-chlorobenzyl)-7-fluoro-5-(methanesulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]acetic        acid or a pharmaceutically acceptable salt thereof, i.e.:

(v) Compounds disclosed in patent application WO2004103970:

-   -   and pharmaceutically acceptable salts thereof, wherein m is 1 or        2, and R₁ is C₁-C₃ alkyl optionally substituted with 1 to 5        halogen atoms.    -   Said compound of formula V is in particular selected from the        group consisting of:

-   [(3R)-4-[(1S)-1-(4-chlorophenyl)ethyl]-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]    acetic acid and pharmaceutically acceptable salts thereof,

-   [(1R)-9-[(1S)-1-(4-chlorophenyl)ethyl]-6-fluoro-8-(methylsulfonyl)-2,3,4,9-tetrahydro-1H-carbazol-1-yl]    acetic acid and pharmaceutically acceptable salts thereof,

-   [(1R)-9-[(1R)-1-(4-chlorophenyl)-2-fluoroethyl]-6-fluoro-8-(methylsulfonyl)-2,3,4,9-tetrahydro-1H-carbazol-1-yl]    acetic acid and pharmaceutically acceptable salts thereof, and

-   [(1R)-9-[(1R)-1-(4-chlorophenyl)-2,2-difluoroethyl]-6-fluoro-8-(methylsulfonyl)-2,3,4,9-tetrahydro-1H-carbazol-1-yl]    acetic acid and pharmaceutically acceptable salts thereof.

(vi)[2-(oxazol-2-yl)-5-(4-{4-[(propan-2-yl)oxy]phenylsulfonyl}piperazin-1-yl)phenoxy]aceticacid

or a pharmaceutically acceptable salt thereof, as described inEP2762141. This compound can be synthesized in accordance with a knownmethod, for example, a method as described in WO 2007/037187 or WO2008/123349.

Exemplary PTGDR-1 antagonists include, but are not limited to, compoundsdescribed as having PGD2 antagonizing activity in PCT PublishedApplications WO97/00853, WO98/25919, WO01/66520, WO02/094830,WO03/022814, WO03/078409, and WO2004/103370; European PatentApplications EP945450, EP944614, and EP 1305286; and U.S. ApplicationPubl. No. 20040220237, 20070244107, and 20080194600, all of which arehereby incorporated by reference in their entirety.

According other embodiments, the PTGDR-1 antagonist is biologicalmolecule antagonist.

For instance said PTGDR-1 antagonist is an antibody, preferablymonoclonal antibody, or antibody fragment, or aptamer directed againstPTGDR-1. Methods of producing polyclonal or monoclonal antibodies arereadily available to the skilled person.

Said PTGDR-1 antagonist may also be an antisense, or interfering RNAinhibiting PTGDR-1 expression.

In an embodiment said PTGDR-1 antagonist is an anti-senseoligonucleotide, in particular an anti-sense oligonucleotide comprisingor consisting of one of the sequence SEQ ID NO:4415 to SEQ ID No:5483,as disclosed in the international patent application published asWO02/081628.

PTGDR-2 Antagonists

According to an embodiment, said PTGDR-2 antagonist is a small moleculeantagonist.

Numerous PTGDR-1 antagonists have been described in the art.

In some embodiments, a PTGDR-2 antagonist is selected from the groupconsisting of:

(i) Compounds disclosed in WO2013088109:

wherein

R1 is C1-C6 alkyl;

R2 is halogen;

R3 is aryl or heteroaryl optionally substituted with one or moresubstituents selected from halo, OH, CN, R6, COR6, CH2R6, OR6, SR6,SO₂R6, or SO₂YR6;

R6 is C1-C6 alkyl, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl,any of which may optionally be substituted with one or more substituentsselected from halo, OH, CN, NO2, C1-C6 alkyl, or O(C1-C6 alkyl); and

Y is NH or a straight or branched C1-C4 alkylene chain;

R4 is H or C1-C4 alkyl; and

R5 is hydrogen, C1-C6 alkyl, aryl, (CH2)_(m)OC(═O)C1-C6 alkyl,((CH2)_(m)O)_(n)CH2CH2X, (CH2)_(m)N(R7)₂, or CH((CH2)_(m)O(C═O)R8)₂;

m is 1 or 2;

n is 1-4;

X is OR7 or N(R7)2;

R7 is hydrogen or methyl;

R8 is C1-C,8 alkyl;

or a pharmaceutically acceptable salt, hydrate, solvate, or complexthereof,

(ii) cycloalkano(1,2-b)indole-sulfonamides as described in EP0242518B1

-   -   wherein R1 is H, fluorine, methyl, methoxy, benzyloxy, or        hydroxyl,    -   R2 is phenyl which is substituted by fluorine, chlorine,        trifluoromethyl, methyl, ethyl, propyl, isopropyl, or methoxy,        and    -   Y is 0 or 1,    -   or pharmaceutically acceptable salts thereof.    -   In particular, a compound of formula VIII is selected from the        group consisting of:

-   Ramatroban    ((R)-3-[[(4-fluorophenyl)sulphonyl]amino]-1,2,3,4-tetrahydro-9H-carbazole-9-propanoic    acid):

or a themodynamically stable form thereof as described in EP1051398,

-   -   and analogs of Ramatroban, in particular TM30642        (3-{3-[(4-fluoro-benzenesulfonyl)-methyl-amino]1,2,3,4-tetrahydro-carbazol-9-yl        {-propionic acid):

TM30643([3-(4-fluoro-benzenesulfo-nylamino)-1,2,3,4-tetrahydro-carbazol-9-yl]-aceticacid):

and

TM30089 (also called CAY10471) (CAS 627865-18-3:2-[3-[(4-fluorophenyl)sulfonyl-methylamino]-1,2,3,4-tetrahydrocarbazol-9-yl]aceticacid):

(iii) OC000549(5-fluoro-2-methyl-3-(2-quinolinylmethyl)-1H-indole-1-acetic acid)

(iv) Setipiprant

and

AZD1981(4-(acetylamino)-3-[(4-chlorophenyl)thio]-2-methyl-1H-indole-1-aceticacid).

Additional PTGDR-2 antagonists can be found in the followingpublications: EP1, 170,594, EP1,435,356 WO2003/066046, WO2003/066047,WO2003/097042, WO2003/097598, WO2003/101961, WO2003/101981,WO2004/007451, WO2004/032848, WO2004/035543, WO2004/106302,WO2005/019171, WO2005/054232, WO2005/018529, WO2005/040112, GB2,407,318,WO2005/040114, WO2005/044260, WO2005/095397, WO2005/100321,WO2005/102338, WO2005/123731, WO2006/034419, WO2006/095183,WO2007/107772, WO2008024746, U.S. Pat. No. 7,405,215, each of which ishereby incorporated by reference in its entirety.

According other embodiments, the PTGDR-2 antagonist is biologicalmolecule antagonist.

For instance said PTGDR-2 antagonist is an antibody, preferablymonoclonal antibody, or antibody fragment, or aptamer directed againstPTGDR-2. In an embodiment said PTGDR-2 antagonist is an antibodydepleting PTGDR-2 expressing cells such as described in theinternational patent application WO2014144865.

Said PTGDR-2 antagonist may also be an antisense, or interfering RNAinhibiting PTGDR-2 expression.

Dual PTGDR-1/PTGDR-2 Antagonists

As used herein, a dual PTGDR-1/PTGDR-2 antagonist is an antagonist forwhich the ratio of antagonist activity against PTGDR-1 and PTGDR-2,respectively, is from 1:10 to 10:1, in particular from 1:50 to 50:1,preferably from 1:100 to 100:1, still preferably from 1:250 to 250:1.

Dual PTGDR-1/PTGDR-2 antagonists that can be used in the frame of theinvention include, for instance, the compounds described in patentapplication WO 2009/085177:

and pharmaceutically acceptable salts thereof,

wherein R1 is alkyl or cycloalkyl; R2 is halo, alkyl, haloalkyl, alkoxy,haloalkoxy or cycloalkyl; and X is chloro or fluoro.

Preferably, said compound of formula VI is AMG 853(2-(4-(4-(tert-butylcarbamoyl)-2-(2-chloro-4-cyclopropylphenylsulfonamido)phenoxy)-5-chloro-2-fluorophenyl)acetic acid):

AMG 853, as well as a method of preparing this compound, are alsodescribed in Liu et al., ACS Med Chem Lett. 2011 Mar. 2; 2(5):326-30.

However, in an embodiment the dual PTGDR-1/PTGDR-2 antagonists offormula VI are excluded from the scope of the invention.

Characterisation of the antagonistic activity of a molecule towardsPTGDR-1 and/or PTGDR-2 can be performed, for instance, by methodsdescribed in Liu et al., ACS Med Chem Lett. 2011 Mar. 2; 2(5):326-30.These methods include:

(i) Determining the molecule's IC₅₀ towards PTGDR-1 and/or PTGDR-2. Thiscan be performed for instance on HEK-293 cells stably expressing humanPTGDR-1 or PTGDR-2. To measure binding, detectably labelled PGD₂, e.g.[³H]-PGD₂, is incubated together with HEK-293 (PTGDR-1 or PTGDR-2) cellsin the presence of increasing concentrations of the molecule to beassayed. After incubation, cells are washed, and the amount of labelledPGD₂ that remained bound to the cells is measured by an appropriatemethod (for instance scintillation counting if [³H]-PGD₂), and theconcentration of compounds required to achieve a 50% inhibition of[³H]-PGD2 binding (the IC₅₀) was determined. The binding buffer containseither 0.5% BSA (buffer binding) or 50% human plasma (plasma binding).

(ii) Determining the molecule's affinity for PTGDR-1. This can beperformed for instance on whole blood drawn into acid-citrate-dextrosevacutainer tubes, treated with test molecule or DMSO, and thenstimulated with a dose response of PGD₂. Cells are then lysed, and cAMPis measured using a competitive ELISA. Comparison of the dose responseto PGD₂ in samples containing DMSO only and samples containing with testmolecule is used in determining K_(b) using the Schild equation (SchildHO. PA2, a new scale for the measurement of drug antagonism. Brit JPharmacol 1947; 2:189-206).

(i) Determining the molecule's affinity for PTGDR-2. This can beperformed for instance on whole blood drawn into acid-citrate-dextroseanticoagulated tubes, treated with test molecule or DMSO, and thenstimulated with a dose response of PGD₂. Fluorochrome conjugatedantibodies are used to label PTGDR-2 positive granulocytes, and PTGDR-2receptor internalization is monitored by flow cytometry. The K_(b) isdetermined using the Schild equation.

Mono- or Combination Treatment

Since both PTGDRs can synergize, antagonizing both PTGDR-1 and PTGDR-2is an advantageous therapeutic modality for treating and/or preventinglupus. Furthermore, this should avoid any risk of potentiation of PGD₂effect on basophils and other cells via the unblocked PTGDR, if only oneof the two PGD₂ receptors is targeted.

According to an embodiment, said PTGDR-1 (or PTGDR-2 antagonist) is adual PTGDR-1/PTGDR-2 antagonist.

According to another embodiment, said PTGDR-1 antagonist is not a dualPTGDR-1/PTGDR-2 antagonist. In said embodiment, the PTGDR-1 antagonistis used either as the sole active ingredient (mono-treatment) for theprevention and/or treatment of SLE, or in combination with at least aPTGDR-2 antagonist (which is neither a dual PTGDR-1/PTGDR-2 antagonist)for the prevention and/or treatment of SLE.

According to another embodiment, said PTGDR-2 antagonist is not a dualPTGDR-1/PTGDR-2 antagonist. In said embodiment, the PTGDR-2 antagonistis used either as the sole active ingredient (mono-treatment) for theprevention and/or treatment of SLE, or in combination with at least aPTGDR-1 antagonist (which is neither a dual PTGDR-1/PTGDR-2 antagonist)for the prevention and/or treatment of SLE.

Additionally, in an embodiment, the PTGDR-1 antagonist, the PTGDR-2antagonist, or the combination of PTGDR-1 antagonist and PTGDR-2antagonist, is used in combination with any other standard therapy forthe treatment of lupus. Therapeutics useful for the treatment of lupusinclude, but are not limited to, nonsteroidal anti-inflammatory drugs(NSAIDs), hydroxychloroquine, corticosteroids, cyclophosphamide,azthioprine, methotrexate, mycophenolate mofetil (MMF) (CelleCept®),belimumab, dehydroepiandrosterone, and rituximab.

The PTGDR-1 and/or PTGDR-2 antagonist(s), and optionally further thestandard therapeutic for the treatment of lupus, are formulated into oneor more pharmaceutical composition(s) comprising said PTGDR-1 and/orPTGDR-2 antagonist(s), and optionally further the standard therapeuticfor the treatment of lupus, and at least one carrier, excipient ordiluent.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier, excipientor diluent refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.

Pharmaceutically acceptable carriers and excipient that may be used inthe compositions of this invention include, but are not limited to, ionexchangers, alumina, aluminium stearate, lecithin, self-emulsifying drugdelivery systems (SEDDS) such as d-a-tocopherol polyethyleneglycol 1000succinate, surfactants used in pharmaceutical dosage forms such asTweens or other similar polymeric delivery matrices, serum proteins,such as human serum albumin, buffer substances such as phosphates,glycine, sorbic acid, potassium sorbate, partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts or electrolytes, such asprotamine sulfate, disodium hydrogen phosphate, potassium hydrogenphosphate, sodium chloride, zinc salts, colloidal silica, magnesiumtrisilicate, polyvinyl pyrrolidone, cellulose-based substances,polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycoland wool fat.

As appreciated by skilled artisans, the pharmaceutical composition issuitably formulated to be compatible with the intended route ofadministration. Examples of suitable routes of administration includetopical route, oral route, intranasal route, intraocular route,parenteral route, including for instance intramuscular, subcutaneous,intravenous, intraperitoneal or local injections. The oral route can beused, provided that the composition is in a form suitable for oraladministration, able to protect the active principle from the gastricand intestinal enzymes.

Preferably, the pharmaceutical composition contains carriers that arepharmaceutically acceptable for an injectable formulation. They may inparticular be sterile, isotonic, saline solutions (monosodium phosphate,disodium phosphate, sodium chloride, potassium chloride, calciumchloride or magnesium chloride etc., or mixtures of such salts), or dry,in particular lyophilized, compositions which by means of the addition,as appropriate, of sterilized water or physiological saline, can forminjectable solutes.

For combined therapy, (i) the PTGDR-1 antagonist and PTGDR-2 antagonist,(ii) the PTGDR-1 antagonist and the standard therapeutic for thetreatment of lupus, (iii) the PTGDR-2 antagonist and the standardtherapeutic for the treatment of lupus, or (iv) the PTGDR-1 antagonist,PTGDR-2 antagonist and the standard therapeutic for the treatment oflupus, are formulated in a single pharmaceutical composition, or saidPTGDR-1 antagonist, PTGDR-2 antagonist, and standard therapeutic for thetreatment of lupus, are formulated in separate pharmaceuticalcompositions for simultaneous use, separate use, or use spread overtime.

Thus the invention also provides for a pharmaceutical compositioncomprising a PTGDR-1 antagonist and a PTGDR-2 antagonist; or a dualPTGDR-1/PTGDR-2 antagonist; a PTGDR-1 antagonist and a standardtherapeutic for the treatment of lupus; a PTGDR-2 antagonist and astandard therapeutic for the treatment of lupus; a PTGDR-1 antagonist, aPTGDR-2 antagonist, and a standard therapeutic for the treatment oflupus; or a dual PTGDR-1/PTGDR-2 antagonist and a standard therapeuticfor the treatment of lupus, for use in a method for preventing and/ortreating SLE.

Accordingly, it is provided a method for preventing and/or treating SLEin a patient in need thereof, wherein said method comprisesadministering said patient with a PTGDR-1 antagonist and a PTGDR-2antagonist, or a dual PTGDR-1/PTGDR-2 antagonist. In an embodiment ofsaid method a standard therapeutic for the treatment of lupus is furtherstep administered with the PTGDR-1 antagonist and PTGDR-2 antagonist, orwith the dual PTGDR-1/PTGDR-2 antagonist. A method for preventing and/ortreating SLE in a patient in need thereof, wherein said method comprisesadministering said patient with a PTGDR-1 antagonist or a PTGDR-2antagonist, and with a standard therapeutic for the treatment of lupus,also makes part of the invention.

The invention also relates to PTGDR-1 antagonist and a PTGDR-2antagonist; or a dual PTGDR-1/PTGDR-2 antagonist; a PTGDR-1 antagonistand a standard therapeutic for the treatment of lupus; a PTGDR-2antagonist and a standard therapeutic for the treatment of lupus; aPTGDR-1 antagonist, a PTGDR-2 antagonist, and a standard therapeutic forthe treatment of lupus; or a dual PTGDR-1/PTGDR-2 antagonist and astandard therapeutic for the treatment of lupus, for use as a combinedpreparation for simultaneous use, separate use, or use spread over time,in a method for preventing and/or treating SLE.

The methods of preventing and/or treating SLE according to the inventionpreferably use an effective amount of PTGDR-1 and/or PTGDR-2 antagonistand/or standard therapeutic for the treatment of lupus.

The expression “effective amount” is intended to mean an amountsufficient to prevent and/or treat a given disease. It will beappreciated that this amount will vary with the effectiveness oftherapeutic agent(s) employed, with the nature of any carrier used, withthe seriousness of the disease and the age of the patient. Thedetermination of appropriate amounts for any given composition is withinthe skill in the art, through standard series of tests designed toassess appropriate therapeutic levels.

According to invention the PTGDR-1 antagonist and/or PTGDR-2 antagonistprevent(s) basophil homing to secondary lymphoid organs.

In the frame of the invention, the PTGDR-1 antagonist and/or PTGDR-2antagonist prevents, limits the extent or reduces the increase inautoantibody titers and/or the occurrence of SLE flares. Alternativelyor furthermore, the PTGDR-1 antagonist and/or PTGDR-2 antagonist alsoprevents, limits the extent or reduces the occurrence organ damages, inparticular to kidneys, heart, lungs, and brain. In an embodiment, thePTGDR-1 antagonist and/or PTGDR-2 antagonist prevents, limits the extentor reduces the occurrence of lupus nephritis.

The invention will be further illustrated in view of the followingfigures and examples.

FIGURES

FIG. 1. Human blood basophil gating strategy and activation status infunction of SLE disease activity.

(a) Flow cytometric analysis of CD203c levels on blood basophils fromsubjects with inactive, mild or active SLE (n=60/40/82, respectively)compared to controls (CT, n=100). (b) Flow cytometric analysis of CD62Llevels on blood basophils from subjects with inactive, mild or activeSLE (n=43/33/66, respectively) compared to controls (CT, n=90). (c) Flowcytometric analysis of CD63 levels on blood basophils from subjects withinactive, mild or active SLE (n=14/6/12, respectively) compared tocontrols (CT, n=12). (a-c) Data are normalized to controls' mean andexpressed in arbitrary units as means+s.e.m. Statistical analyses wereby Mann-Whitney tests. NS: not significant, *: P<0.05, **: P<0.01, ***:P<0.001.

FIG. 2. Basopenia and basophil activation status correlate with diseaseactivity and are specific for lupus nephritis among other active renaldiseases. (a) Blood basophils per μL as determined by flow cytometryfrom healthy controls (CT) and subjects with inactive, mild or activeSLE (n=87/58/38/84, respectively) ad defined in the online methods. (b)Spearman correlation between blood basophil numbers and SLEDAI(r=−0.3629, p<0.0001) shown on a linear scale. (c) Proportion of bloodHLA-DR⁺ basophils as determined by flow cytometry healthy controls (CT)and subjects with inactive, mild or active SLE (n=96/60/40/84,respectively). (d) Receiver-Operating Characteristic (ROC) analysis ofthe proportion of HLA-DR⁺ basophils in SLE patients (n=184) versuscontrols (n=97) (thick line, AUC=0.9091) and of dsDNA-specific IgGtiters in SLE patients (n=123) versus controls (n=39) (dotted line,AUC=0.8384). (e) Blood basophils per μL as in (a) from subjects with thefollowing active renal diseases: IgA-N: IgA nephropathy; HSP:Henoch-Schönlein purpura nephropathy; DN: Diabetic Nephropathy; MN:membranous nephropathy; OGN: Other glomerular nephropathies; NGKD:Non-Glomerular Kidney Diseases; aLN: active Lupus Nephritis; compared tohealthy controls (n=40/20/39/22/42/46/81/87, respectively). (f)Proportion of blood HLA-DR⁺ basophils in patients with active renaldiseases as in (e) (n=40/20/39/21/51/47/77, respectively) compared tocontrols (n=96). (a,c,e,f) Data are expressed as means+s.e.m.Statistical analyses were by Mann-Whitney tests. Comparison to healthycontrols' median is shown above each bar and to the corresponding barswhen indicated. NS: not significant, *: P<0.05, **: P<0.01, ***:P<0.001, ****: P<0.0001.

FIG. 3. PGD₂/PTGDRs and CXCL12/CXCR4 axes contribute to SLE patientspecific basophil phenotype

(a) Flow cytometric analysis of PTGDR-2 (CRTH2) levels on bloodbasophils from healthy controls (CT) and subjects with inactive, mild oractive SLE (n=71/48/31/60, respectively). (b) 11β-prostaglandin F₂α(11β-PGF₂α) levels in plasma from controls and individuals withinactive, mild or active SLE (n=29/31/19/37, respectively) as measuredby EIA. (c) Blood basophils per μl of blood in subjects with SLEclassified on the basis of low (n=51) or high (n=34) 11β-PGF₂α plasmalevels (titer below or above CT titer mean+2 standard deviations,respectively). (d) Flow cytometric analysis of CXCR4 levels on bloodbasophils from CT and subjects with inactive, mild or active SLE(n=66/32/20/51, respectively). (e) CXCL12 levels in plasma from controlsand individuals with inactive, mild or active SLE (n=63/43/29/59,respectively) as measured by ELISA. (f) Flow cytometric analysis of thelevels of CD164 on blood basophils from CT and subjects with inactive,mild or active SLE (n=33/15/7/26, respectively). (a,d,f) Data arenormalized to the mean of CT values and expressed in arbitrary units.(a-f), Statistical analyses were by Mann-Whitney tests. Comparison tohealthy controls' median is shown above each bar and to thecorresponding bars when indicated. Data are expressed as means+s.e.m.NS: not significant, *: P<0.05, **: P<0.01, ***: P<0.001, ****:P<0.0001.

FIG. 4. Associations between plasma 11β-PGF2α & CXCL12 titers, basophilCXCR4 & CD164 expression levels and lupus specific basopenia

(a) Spearman correlation between blood basophil number per mL of bloodand 11β-PGF2α plasma levels (r=−0.2585, P=0.0169, n=85) shown on alogarithmic scale. (b) Spearman correlation between blood basophilnumber per mL of blood and relative CXCR4 levels on basophils (asdefined in FIG. 2d ) (r=−0.4692, P<0.0001, n=101) shown on a logarithmicscale. (c) Blood basophils per μl of blood in subjects with active SLEclassified on the basis of low or high CXCL12 plasma levels (titer belowor above control titer mean+2 standard deviations, respectively). Dataare expressed as means+s.e.m. Statistical analysis was by Mann-Whitneytest. *: P<0.05. (d) Spearman correlation between blood basophil numberper mL of blood and relative CD164 levels on basophils (as defined inFIG. 2f ) (r=−0.4165, P=0.0029, n=49) shown on a logarithmic scale.

FIG. 5. CXCR4-mediated basophil migration ex vivo and in vivo isenhanced by PGD₂

(a) Migration assays of human blood basophils from healthy controls (CT,n=6) and SLE patients (n=6) towards a CXCL12 gradient. (b) Migrationassays of human blood basophils towards IL-3, CCL3, CCL5, CXCL2 and PGD2gradients from healthy controls (Controls, n=8/4/3/4/7, respectively)and from SLE patients (SLE, n=6/3/6/3/5, respectively). (c) RelativedsDNA-specific IgE levels in plasma from inactive, mild or active SLEindividuals (n=41/29/51, respectively) normalized to the control valuesmean (n=38) as measured by ELISA. (a-c) Statistical analyses were byMann-Whitney tests compared to CT. (d) CXCR4 expression levels on bloodbasophils after 18 hours of incubation without (−) or with (+) PGD₂,mouse anti-human IgE or IL-3 was assessed by flow cytometry. (e)Migration assays of human blood basophils stimulated as in (d) towards aCXCL12 gradient. (d,e) Statistical analyses were by paired Student ttest. (f) Expression level variation of the indicated basophil markersbetween peritoneal and blood basophils from patients undergoingperitoneal dialysis and being treated for non-sterile peritonitis (n=6)were assessed by flow cytometry. Statistical analysis was by one samplet test compared to a 0 theoretical value. (a,b,e) Corrected migration asdescribed in the methods. (a-f) Data are expressed as means±s.e.m. NS:not significant, #: P=0.06, *: P<0.05, **: P<0.01, ***: P<0.001, ****:P<0.0001.

FIG. 6. Suboptimal IgE-mediated basophil activation leads to increasedPTGDR-2 expression on human basophils.

PTGDR-2 (CRTH2) expression levels (as defined in online methods) onblood basophils from healthy donors after 18 hours of incubation without(−) or with (+) PGD2, mouse anti-human IgE or IL-3. Statistical analyseswere by paired Student t test.

FIG. 7. PGD₂ is sufficient to enhance the CXCL12-dependent basophilhoming to SLOs occurring in lupus-prone mice

(a) Ex vivo migration of basophils from whole WT or Lyn^(−/−)splenocytes to CXCL12. (b) CXCR4 expression levels on basophils from theindicated compartments in aged WT (n=16) and Lyn^(−/−) (n=14) animals.Data are normalized to the mean CXCR4 expression level of WT bloodbasophils. Statistical analyses placed directly above each bar comparedthe value for one given compartment to the blood compartment of thecorresponding genotype. Statistical analyses between both genotypes foreach compartment are also indicated. (c) Basophil number per peritoneumin young WT mice 24 hours after intraperitoneal (ip) injection of PBS orCXCL12 and compared to steady state (−) values (n=13/15/5,respectively). (d) CXCR4 expression levels on basophils from mesentericlymph nodes (mLN) of young Lyn^(−/−) mice 24 hours after PBS or PGD₂ ipinjection normalized to PBS injected mice values' mean. (e) Basophilnumber in mLN of young Lyn^(−/−) mice 24 hours after ip injection of theindicated compound(s). (f) CXCR4 expression levels on spleen basophilsfrom 15 weeks old WT mice incubated ex vivo for 24 hours with theindicated compound and normalized to the control values' mean. (a-f)Data are expressed as means±s.e.m. Basophils number and CXCR4 expressionwere assessed by flow cytometry. Statistical analyses were by unpaired ttest with Welch's correction (a-e) and by paired Student t test (f). NS:not significant, #: P=0.058, *: P<0.05, **: P<0.01, ***: P<0.001, ****:P<0.0001.

FIG. 8. CXCL12 or PGD2 ip injection in mice induce a CXCR4-dependentbasophil accumulation in SLOs and peritoneum.

(a) Proportion of basophil (×103) among living CD45+ cells in mesentericlymph node (mLN) of young WT mice 24 hours after intraperitoneal (ip)injection of PBS (n=13) or CXCL12 (n=15) and compared to steady state(−) values (n=5). (b) Basophil number in spleen of young Lyn−/− mice 24hours after ip injection of the indicated compound(s). (c) Basophilnumber in peritoneum of Lyn−/− mice 24 hours after ip injection of theindicated compound(s). Data are expressed as means±s.e.m. Statisticalanalyses were by unpaired Student t test with Welch's correction. NS:not significant, *: P<0.05, **: P<0.01, ***: P<0.001.

FIG. 9: cAMP and PTGDRs specific agonist effects on CXCR4 expression bymouse spleen basophils ex vivo.

(a-b) Relative CXCR4 expression levels on basophils (defined as CD19⁻TCRβ⁻ CD3⁻ CD49b⁺ FcεRIα⁺ CD123⁺ CD45^(lo)) and T cells (defined asCD45⁺CD3⁺TCRβ⁺ cells) in splenocytes incubated 4 hours without (−) orwith the indicated concentration of N6,2′-O-dibutyryl-adenosine3′:5′-cyclic monophosphate (db-cAMP) or 1 μM PGD₂ as determined by flowcytometry. Statistical analyses were by paired Student t tests. NS: notsignificant, *: P<0.05, **: P<0.01, ***: P<0.001. (c) Relative CXCR4expression levels on spleen basophils (defined as in (a)) incubated 4hours without (O) or with the indicated concentration (nM) of theindicated compound(s). DK-PGD₂: 13,14-dihydro-15-keto-PGD₂ (PTGDR-2specific agonist); BW245c:3-(3-Cyclohexyl-3-hydroxypropyl)-2,5-dioxo-(R*,S*)-(±)-4-imidazolineheptaniocacid (PTGDR-1 specific agonist) as determined by flow cytometry.Statistical analysis was by two-ways ANOVA followed by a Tukey'smultiple comparisons test. (a-c) Data are normalized to control valuemean (per group, n=4 to 8). Data are expressed as mean+s.e.m. Allexperiments were realized with splenocytes from 8-12 weeks old WT mice.

FIG. 10. Blockade of basophil accumulation in SLOs dampens lupus-likedisease activity

(a,b) Proportion of basophils (CD19⁻TCRβ⁻CD49b⁺FcεRIα⁺CD123⁺CD45^(lo))among singlets living CD45⁺ cells in mesenteric (a) and other (cervical,brachial and inguinal) lymph nodes (b) from 10 to 12 weeks-old Lyn^(−/−)mice injected over ten days with PBS (open circles) or PGD₂ alone (opensquares), and PGD₂ injected and basophil depleted (MAR-1) (opendiamonds) or not (IgG) (open triangles) as described in the methods. (c)IA-IE expression levels on LN basophils from mice as in (b). NA: notapplicable. (d,e) Proportion of short lived plasma cells CD19⁺CD138⁺among singlets living CD45⁺ cells in spleen (d) and lymph nodes (e) inthe same mice as in (b). (f) Representative immunofluorescence stainingfor C3 and IgG deposits in kidneys from mice as indicated in (b) (scalebar=1 mm) and their corresponding quantifications in PBS (n=3), PGD₂(n=4), PGD₂+control IgG (n=3) and PGD₂+MAR-1 (n=3) injected mice. (g)Fold increase in urine albumin/creatinine ratio before and after PBS orPGD₂ 10 days-long treatment. (a-e) Basophil, plasma cell numbers andIA-IE expression levels were assessed by flow cytometry. (a-g) Data areexpressed as means±s.e.m. Statistical analyses were by unpaired studentt tests. #: P=0.0571, *: P<0.05, **: P<0.01, ***: P<0.001, ****:P<0.0001. One representative out of three independent experiments isshown.

FIG. 11. Blockade of basophil accumulation in SLOs dampens lupus-likedisease activity

Quantifications of immunofluorescence staining for C3 and IgG depositsin kidneys from aged Lyn^(−/−) mice treated (n=8) or not (vehicle, n=9)with PTGDR-1 and PTGDR-2 antagonists for ten days.

FIG. 12. Targeting PTGDRs blocks CXCR4-mediated basophil accumulation inSLOs and reduces the lupus-like disease activity.

(a-h) Comparisons between aged wild-type (WT) and Lyn^(−/−) mice treatedor not (vehicle) for 10 days with PTGDR-1 and PTGDR-2 antagonists asdescribed in the online methods. (a,b,c) Flow cytometric analysis ofbasophils among living CD45⁺ cells in lymph nodes (cervical, axillar,inguinal and mesenteric) (a) and spleen (b). (c) CXCR4 expression levelson spleen basophils. (d) Proportion of short lived plasma cellsCD19⁺CD138⁺ among living CD45⁺ cells in lymph nodes as in (a) wasdetermined by flow cytometry. (e) Optical density (O.D.) values at 450nm of dsDNA-specific IgG in plasma from the indicated mice as measuredby ELISA (×10³). (f) Total IgE levels as measured by ELISA in plasmafrom the indicated mice. (g,h) IL-4 (g) and IL-1β (h) concentration inpg per mg of total kidney protein extract from the indicated mice asmeasured by ELISA. (a-h) WT vehicle, n=5; WT treated, n=4; Lyn^(−/−)vehicle, n=8; Lyn^(−/−) treated, n=8. Data are expressed as mean+s.e.m.Statistical analyses were by unpaired Student t tests. NS: notsignificant, *: P<0.05, **: P<0.01.

FIG. 13. Treatment with PTGDRs antagonists reduces specifically basophilaccumulation in SLOs leading to reduced short-lived plasma cell numberand serum ANA titers.

(a-d) Comparisons between aged wild-type (WT) and Lyn−/− mice treated ornot (vehicle) with PTGDR-1 and PTGDR-2 antagonists for ten days. (a,b)Flow cytometric analysis of basophil proportion among CD45+ bone marrow(BM) cells (a) and blood leukocytes (b). (c) Flow cytometric analysis ofshort lived plasma cells CD19+CD138+ among living CD45+ cells. (d)Anti-nuclear antibodies (ANA) levels as measured by ELISA in plasma fromthe indicated mice. (a-d) WT vehicle, n=5; WT treated, n=4; Lyn−/−vehicle, n=8; Lyn−/− treated, n=8. Data are expressed as means+s.e.m.(a-c) Statistical analyses were by unpaired Student t test. (d)Statistical analysis was by Mann-Whitney test. NS: not significant, *:P<0.05, **: P<0.01.

FIG. 14. Graphical abstract

In systemic lupus erythematosus (SLE), a loss of self-tolerance inducesthe expansion of autoreactive (AR) T and B cells. Autoreactive plasmacells secrete autoreactive antibodies which will bind self-antigens ofnuclear origin and complement factors to form circulating immunecomplexes (C10). The deposition of these CIC or autoreactive antibodiesin target organs is associated with local lesions, inflammation (andPGD₂ production), and organ damages. Healthy basophils can get activatedby the binding of CIC to Fc receptors (FcεRI and FcγRs) to express moreprostaglandin D₂ (PGD₂) receptors (PTGDRs) and activation markers suchas CD203c. As chronic inflammation settles, so does the secretion ofvarious inflammatory mediators in blood, including PGD₂. PGD₂ issufficient to induce PGD₂ production by circulating basophils themselvesleading to an autocrine effect of PGD₂. This leads to an increasedsurface expression of CXCR4 and enable basophil sensitivity to CXCL12gradients. As a result, basophils are more eager to migrate to SLOs,which are known to secrete more CXCL12 during lupus pathogenesis. There,basophils support autoreactive T and B cells through their expression ofactivating molecules such as mBAFF, MHC-II or the secretion of variouscytokines such as IL-4 and IL-6. Moreover, basophils can promoteautoreactive antibody production and IgE class switching of B cells. AsCIC and autoreactive IgE titers increase, so will targeted organinflammation, PGD₂ and CXCL12 titers and basophils homing to SLOs. It isassumed that basophils drive an amplification loop of the disease andblocking their recruitment to SLOs would prevent rise in autoantibodytiters and consequent flares.

EXAMPLES Example 1: Materials and Methods

Mice.

C57BL/6J wild-type (WT) mice were purchased from Charles RiverLaboratories (L'Arbresle, France) and Lyn^(−/−) mice on a pure C57BL/6background were bred in our animal facility. For lupus-like diseasestudies, mice were aged for a minimum of 40 weeks before treatment andanalysis. For other ex vivo or in vivo analysis, young mice were between8 and 12 weeks old, unless otherwise specified. Mice were maintained inspecific pathogen-free conditions, used in accordance with French andEuropean guidelines and approved by local ethical committee and by theDepartment of Research of the French government under the animal studyproposal 02484.01.

Patients.

Blood samples were collected from adult subjects enrolled in aprospective long term study of systemic lupus erythematosus (SLE) andchronic renal diseases. The study was approved by the Comité Régional deProtection des Personnes (CRPP, Paris, France) under the referenceID-RCB 2014-A00809-38. Diagnostics of inpatients were not known by theinvestigators at the time of sample processing and flow cytometryanalysis. SLE samples were obtained from in- and outpatients andclinical data were harvested after approval by the Comission Nationalede l'Informatique et des Libertés (CNIL). All SLE subjects fulfilled theAmerican College of Rheumatology classification criteria for SLE. SLEand healthy control (HC) donor characteristics are shown in Table 1(below). Lupus activity was assessed by SELENA-SLEDAI (Safety ofEstrogens in Lupus Erythematosus National Assessment—SLE DiseaseActivity Index) scores. Based on the SLEDAI score, lupus activity wasclassified as inactive (0-1), mild (2-4) and active (>4). All sampleswere collected in heparin blood collection tubes and processed within 4hours. A written informed consent was obtained from all subjects. Activelupus nephritis subjects were defined by histologically active classesIII or IV+/−V nephritis, in accordance with the ISN/RPS classification(Weening, J. J., et al., J. Am. Soc. Nephrol. 15, 241-250 (2004)).

Antibodies and Flow Cytometry

All antibodies were from commercial sources. Flow cytometry acquisitionwas done with a LSRII Fortessa using DIVA software (BD Biosciences).Blood sample processing procedure was as previously described (Charles,N. et al., Nat. Med. 16, 701-707 (2010)). All data relative to markerexpression levels are expressed as the ratio between the geometric meanfluorescence intensity (Geo MFI) of the indicated marker on the cells ofinterest and the Geo MFI of the corresponding isotype control. Data werenormalized or not as indicated in figure legends. Data analysis wasrealized with FlowJo v.X.0.7 (Treestar).

Chemokines, Cytokines, 11β-PGF_(2α) and Immunoglobulin MeasurementAssays

All commercial assays were performed according to the manufacturerinstructions. 11β-Prostaglandin F_(2α) enzyme immunoassay (EIA) kitswere from Cayman Chemicals (Ann Arbor, Mich.). Mouse ANA enzyme linkedimmunosorbent assay (ELISA) kits were from ADI (San Antonio, Tex.).Human and mice CXCL12 ELISA kits were from R&D Systems (Minneapolis,Minn.). Assessment of cytokine content in the kidney was previouslydescribed (Charles, N. et al., Nat. Med. 16, 701-707 (2010)). Mouse IL4and IL1β ELISA kits were from BioLegend (San Diego, Calif.). Mouse IgEQuantification ELISA kits were from Bethyl Laboratories (Montgomery,Tex.). Human and mouse anti-dsDNA IgG and IgE were quantified aspreviously described (Charles, N. et al., Nat. Med. 16, 701-707 (2010)).Absorbance was assessed by an Infinite 200 Pro plate reader (TECAN,Mannedorf, Switzerland).

Human Basophil Purification and Enrichment.

Human basophils were purified to >95% by negative selection with theHuman Basophils Enrichment kit (Stemcell Technologies, Grenoble, France)for culture, stimulation and chemotaxis experiments. In some chemotaxisexperiments, human basophils were enriched to 3-5% by negative selectionwith the Human PE positive selection kit (Stemcell Technologies) byusing a cocktail of PE-conjugated anti-CD3, CD19 and CD89 (BioLegend).These kits were used following manufacturer instructions.

Imaging Flow Cytometry

Basophils were enriched to 3-5% as described above and frozen at −80° C.in 90% FCS 10% dimethyl sulfoxide until enough samples were collected.Thawed cells were stained, fixed (IC fixation buffer, eBioscience) andpermeabilized (Wash Perm Buffer, BioLegend) following the manufacturers'instructions. Anti-human CXCR4 or its isotype (BioLegend) were used forintracellular staining. DAPI was added prior to cytometry analysis.Basophils were gated as Singlets cells/Focus high/DAPI high/PE⁻ CD123⁺FcεRIα⁺ CD303⁻. CXCR4 expression was determined for each basophil as theratio of the geometric mean of their CXCR4 intensity on the meanbasophil CXCR4 FMO (Fluorescence Minus One) intensity. Internalizationscores were determined using Fc□RI□ staining as a membrane marker andCXCR4 staining as the probe. For each sample, externalization scorecorresponds to [1−internalization score]. All analyses were performedusing the ImageStream X Mark II imaging flow cytometer and the IDEAS v6software (AMNIS).

Basophil Culture and Stimulation

Human basophils and mouse splenocytes were cultured in culture medium(RPMI 1640 with Glutamax and 20 mM HEPES, 1 mM Na-pyruvate,non-essential amino acids 1X (all from Life Technologies, Saint-Aubin,France), 100 μg/ml streptomycin and 100 U/ml penicillin (GE Healthcare,Vélizy, France) and 37.5 μM β-mercaptoethanol (Sigma-Aldrich, MO))supplemented with 20% heat-inactivated fetal calf serum at 37° C. and 5%CO₂. 18 hours-long stimulation prior to migration assays were done inculture medium at 1×10⁶ cells per mL by adding 1 nM of IL-3 (Peprotech),1 μM of prostaglandin D2 (PGD₂), 1 μM of the PTGDR-1 specific agonistBW245c, 1 μM of the PTGDR-2 specific agonist 13,14-dihydro-15-ketoProstaglandin D₂ (DK-PGD₂) (all from Cayman chemicals) or 5 ng/mL ofanti-IgE (mouse anti-human IgE or rat anti-mouse IgE, both from ThermoScientific). All cells were washed twice before any migration assay.Control conditions were always with the same vehicle concentration asstimulated conditions.

For CXCR4 overexpression modulation by PTGDRs antagonists, PGD₂ andH-PGDS inhibitor, purified basophils were resuspended in RPMI containing0.1% BSA+/− the following compounds: vehicle (ethanol 0.1% ∘), 1 μMPTGDR-1 antagonist Laropiprant, 1 μM PTGDR-2 antagonist CAY10471, 1 or10 μM PGD₂, and 1 μM of Prostaglandin D Synthase (hematopoietic-type)Inhibitor I (catalog #16256) (all from Cayman Chemical). Cells wereincubated for 4 hours at 37° C. and 5% CO₂ and surface expression of theindicated markers were assessed by flow cytometry.

Basophil Migration and Apoptosis Assays

Migration assays were performed in culture medium supplemented with 0.1%bovine serum albumin (BSA, Sigma-Aldrich) in Transwell 5 μmpolycarbonate permeable support 6.5 mm inserts (Corning, N.Y., N.Y.) for3 hours at 37° C. and 5% CO₂ with 1×10⁵ purified basophils or 2×10⁵enriched basophils at 1×10⁶ cells per mL. Purified or enriched basophilsfrom the upper and bottom chambers were counted at the end of the assay.Basophil content and phenotype was determined by flow cytometry byanalyzing more than 100 basophils. Purification or enrichment of humanbasophils didn't show any difference in the measured migration for alltested chemokines. Migration was defined as the ratio between the numberof basophils in the bottom chamber and the number of basophils in theupper plus the bottom chambers. Spontaneous migration was defined as themigration observed without any chemokine in the bottom chamber.Corrected migration was defined as the difference between specific andspontaneous migration. For migration assays the following concentrations(known to be optimal) were used for each compound: Human IL-3: 300 pM(Peprotech), human CCL3, CCL5, and CXCL12: 50 nM; CXCL2 (all fromBioLegend) and PGD₂ (Cayman chemicals): 100 nM. Migration to human CCL3,CCL5, CXCL2, and PGD₂ was done in the presence of IL-3 at 300 pM in bothchambers. Migration with IL-3 represents chemokinetism: IL-3 was addedin both chambers to the same concentration and compared to thespontaneous migration observed without IL-3. Effects of 24 hoursincubation with IL-3 or PGD2 (as described above) on basophil apoptosiswere estimated by using the FITC Annexin V Apoptosis Detection Kit fromBD Biosciences and used accordingly to manufacturer's instructions.

In Vivo Experiments

For CXCL12-induced basophil in vivo migration assays, 200 μL of PBScontaining 100 ng of murine CXCL12 or PBS alone were injectedintraperitoneally (ip) in 8-12 weeks old WT mice. For PGD₂-inducedbasophil in vivo migration assays, 100 μL of PBS (with 2 μL of ethanol)alone, or PBS±20 nmoles of PGD₂±200 μg of AMD3100 (all from CaymanChemicals) were injected ip in 8-12 weeks old Lyn^(−/−) mice. In allcases, 24 hours later, mice were euthanized and peritoneal lavage,blood, mesenteric lymph nodes and spleen were collected and prepared forFACS analysis as previously described (Charles, N. et al., Nat. Med. 16,701-707 (2010)). For acceleration of disease development by PGD₂injections, 12 weeks old Lyn^(−/−) mice were injected ip with 20 nmolesof PGD₂ or vehicle in PBS, every two days for 10 days, for a total of 6injections. Mice were analyzed the day following the last injection. Fortreatment with PTGDRs antagonists, aged WT and Lyn^(−/−) mice weretreated by oral doses of 5 mg/kg of Laropiprant and CAY10471 (Caymanchemicals) or equivalent dose of ethanol (vehicle) in tap water twice aday for ten days. Then, mice were euthanized and blood, plasma, spleen,bone marrow, kidneys and lymph nodes (cervical, brachial, inguinal andmesenteric) were analyzed as previously described (Charles, N. et al.,Nat. Med. 16, 701-707 (2010)). The treatment didn't affect weight andcell numbers in the different organs. Cell viability was assessed by theutilization of Ghost Dye Violet 510 (Tonbo, San Diego, Calif.).

Analysis of Glomerular Deposition of IgG and C3, and Kidney Function.

Kidney preparation for immunofluorescence analysis of C3 and IgGdeposits was as previously described (Charles, N et al. Nat. Med., 2010,16, 701-707, 2159). Quantification of C3 and IgG deposits was realizedby using ImageJ software (v1.49p, NIH, USA). A minimum of 20 glomeruliwas quantified per kidney. For assessment of kidney function thealbumin/creatinine ratio (ACR) was determined. Urine was collected fromeach mouse before and after treatment. The albumin concentration wasmeasured with a mouse albumin ELISA (Bethyl laboratories, Montgomery,Tex.). A creatinine assay (R&D systems, Minneapolis, Minn.) was used todetermine urine creatinine concentrations. Results are expressed as afold increase corresponding to the ratio of the ACR after/beforetreatment.

Statistical Analysis.

Distribution was assessed with D'Agostino-Pearson omnibus normality testor Kolmogorov-Smirnov test, depending on sample size, to performappropriate analyses. When more than 2 groups were compared, one-wayanalysis of variance (ANOVA) tests were conducted before the indicatedpost-tests when significance (p<0.05) was reached. All tests run weretwo-tailed. Statistics were performed with GraphPad Prism V5 and V6(GraphPad) and with STATA 12 (Statacorp) softwares.

Example 2: Results

Specific SLE and Lupus Nephritis Basophil Phenotype

On a cohort of individuals with SLE (n=188, Table 1), we first validatedthat SLE subject basophils had an activated phenotype as shown byincreased CD203c (a basophil activation marker) and CD62L (L-selectin,involved in leukocyte rolling) expressions as compared to healthycontrol (HC) ones (n=98, FIG. 1a-b , Table 1) (Charles, N. et al., Nat.Med. 16, 701-707 (2010)).

However, SLE basophils did not display a degranulated phenotype (asmeasured by their CD63 expression level, FIG. 1c ). Basopenia appearedto be a good marker of disease correlating with SLE disease activityindex (SLEDAI, American College of Rheumatology Ad Hoc Committee onSystemic Lupus Erythematosus Response, C. The American College ofRheumatology response criteria for systemic lupus erythematosus clinicaltrials: measures of overall disease activity. Arthritis Rheum. 50,3418-3426 (2004)) (Spearman r coefficient=−0.3629, P<0.0001) (FIG. 2a,b), whereas proportion of HLA-DR positive basophils was better (ReceiverOperating Characteristic (ROC) Area Under Curve (AUC)=0.9091) thananti-dsDNA IgG (ROC AUC=0.8384) to discriminate SLE subjects fromhealthy control (HC) (ROC AUC comparison by DeLong method (DeLong, E. R.et al., Biometrics 44, 837-845 (1988)): P=0.03) (FIG. 2c,d ). Moreover,basopenia and high proportion of HLA-DR⁺ basophils were specific markersfor active lupus nephritis when compared to other active renal diseases(FIG. 2e,f ). Of note, these SLE-specific basophil parameters wereindependent of SLE patient treatments at the time of blood harvestingand independent of gender (data not shown). Altogether, these datavalidated that activated basophils, peripheral basopenia and highproportion of HLA-DR⁺ basophils are hallmarks of active SLE individuals.Moreover, our data strongly suggest that lupus environment drives abasophil sub-optimal activation (without a detectable degranulationresponse) and a basophil redistribution to SLOs.

TABLE 1 SLE Patients and control characteristics Lupus patients InactiveMild Active Healthy Variables All SLE (SLEDAI ≦ 1) (1 ≦ SLEDAI ≦ 4)(SLEDAI > 4) controls Demographic characteristics n 188 61 41 86 110Age, mean ± SD, years 37.8 ± 12.3 43.4 ± 14.1 36.1 ± 10.2 34.7 ± 10.634.5 ± 14.9 Female Gender, n (%) 167 (89) 51 (84) 36 (88) 80 (93) 51(47) Lupus characteristics Disease duration, mean ± SD, years 10.5 ±8.2  11.7 ± 9.0  12.1 ± 7.5  9.1 ± 8.0 — Anti-dsDNA Ab positive, n (%)104 (55) 10 (18) 27 (64) 67 (79) — History of lupus nephritis, n (%) 144(77) 36 (59) 29 (66) 79 (92) — SLEDAI Mean ± SD 6.6 ± 7.6 0.0 ± 0.2 2.9± 1.0 13.0 ± 6.8  — Median (range) 4 (0-43) 0 (0-1) 2 (2-4) 12 (5-43) —Treatment characteristics Current prednisone dose (mg/day) Mean ± SD23.6 ± 80.8 4.1 ± 3.9 7.2 ± 8.8  44.8 ± 115.4 — 15 mg/day or higher, n(%) 38 (20) 0 (0) 4 (9) 34 (40) — Concurrent immunosuppressive therapy(n, %) hydroxychloroquine, n (%) 159 (84) 54 (88) 38 (93) 67 (78) —mycophenolate mofetil, n (%) 50 (27) 15 (24) 16 (36) 19 (22) —cyclophosphamide, n (%) 3 (2) 0 (0) 0 (0) 3 (3) — azathioprine, n (%) 26(14) 8 (13) 7 (17) 11 (13) — SLEDAI: Systemic Lupus ErythematosusDisease Activity Index.

PGD₂/PTGDRs and CXCR4/CXCL12 Axes in Basophils from SLE Subjects

To decipher basophil activation and redistribution to SLOs during lupuspathogenesis, we analyzed on basophils from SLE subjects, versus HC, theexpression levels of receptors for chemotactic molecules known to bedysregulated in individuals with lupus or chronic inflammatory diseases(Pellefigues, C. & Charles, N., Curr. Opin. Immunol. 25, 704-711(2013)). Most of the screened receptor expressions were notsignificantly different from the ones observed on HC basophils (Table2). Of note, Thymic Stromal Lymphopoietin Receptor (TSLP-R), IL-33receptor (T1/ST2), C—C motif ligand receptor (CCR) 4, CCR6 and CCR7could not be detected on basophils (Table 2).

However, PTGDR-2 expression was increased on basophils from SLEindividuals (Table 2, FIG. 3a ) as did its ligand titers in their plasma(11β-PGF₂α levels, the main plasmatic PGD₂ metabolite, are presented)(FIG. 3b ). An inverse correlation between 11β-PGF₂α titers and bloodbasophil counts in subjects with SLE was found (Spearman r=−0.2585,P=0.0169) (data not shown). Moreover, high levels of 11β-PGF₂α wereassociated with increased basopenia in SLE subjects (FIG. 3c ).Together, these data strongly suggest that PGD₂ and its receptors areassociated with basophil activation and extravasation during lupus.

CXCR4 expression was increased on basophils from all SLE individuals,but active SLE patients showed an even more marked increase (Table 2,FIG. 3d ). CXCL12 plasma titers followed the same pattern of increase asits receptor did on basophils (FIG. 3e ). Basophil CXCR4 expressionlevels were negatively correlated with blood basophil count in SLEsubjects (Spearman r=−0.4692, P<0.0001) (FIG. 4b ). Moreover, in activeSLE subjects, high CXCL12 titers were associated with a more pronouncedbasopenia (FIG. 4c ). Endolyn (CD164) is a transmembrane syalomucinenhancing sensitivity to CXCL12 when associated to CXCR4 and is alsoknown as a human basophil activation marker. CD164 levels on basophilsfrom active SLE subjects followed the same expression pattern as CXCR4and were correlated with basopenia (Spearman r=−0.4165, P=0.0029),suggesting an increased sensitivity of SLE basophils to CXCL12 in vivo(FIG. 3f and FIG. 4d ). Basophils are known to express CXCR4 mostlyintracellularly. Analyses by imaging flow cytometry showed that SLEpatient basophils had an increased CXCR4 content and that it was moreexternalized than in HC basophils (data not shown).

TABLE 2 Basophil surface marker expression level relative to SLE diseaseactivity Normalized expression levels (to CT mean) on basophils from:Mean (±Se, n, p Mann Whitney test vs CT) Chemokine Healthy or cytokinevolonteers Inactive SLE Mild SLE Active SLE receptor (controls, patientspatients patients analyzed CD# Ligand(s) CT) (SLEDAI ≦ 1) (1 < SLEDAI ≦4) (SLEDAI > 4) CCR1 CD191 CCL3, 5, 1 1.013 1.199 1.11 7, 23 (±0.14, 13,(±0.17, 15, (±0.13, 8, (±0.18, 19, 1) 0.78) 0.40) 1) CCR2 CD192 CCL2, 7,1 1.04 1.03 1.04 8, 13, 16 (±0.11, 30, (±0.15, 19, (±0.25, 14, (±0.2,20, 1) 0.89) 0.57) 0.58) CCR3 CD193 Eotaxin 1 0.8529 0.9268 0.9925(CCL11, (±0.09, 39, (±0.10, 10, (±0.11, 13, (±0.13, 20, 24, 26) 1)0.4642) 0.9663) 0.9681) CCR4 CD194 CCL2, 4, ND ND ND ND 5, 17, 22 CCR5CD195 CCL5, 3, 1 0.7726 1.377 1.031 4, 3L1 (±0.14, 6, (±0.09, 5, (±0.42,3, (±0.19, 7, 1) 0.43) 0.38) 0.94) CCR6 CD196 CCL20 ND ND ND ND CCR7CD197 CCL19, 21 ND ND ND ND CXCR1 CD181 IL-8 1 1.104 1.197 1.187 (±0.12,25, (±0.21, 6, (±0.17, 6, (±0.27, 13, 1) 0.63) 0.28) 0.90) CXCR2 CD182IL-8, 1 1.181 0.9324 1.053 CXCL1, 2, (±0.04, 53, (±0.12, 27, (±0.08, 15,(±0.12, 37, 3, 5 1) 0.85) 0.21) 0.22) CXCR4 CD184 CXCL12 1 1.317**1.279* 2.622*** (±0.04, 66, (±0.09, 32, (±0.11, 20, (±0.42, 51,<0.001) 1) 0.0068) 0.0385) PTGDR-2 CD294 PGD₂ 1 1.404 1.329* 1.328*(CRTH2) (±0.06, 71, (±0.11, 48, (±0.13, 31, (±0.11, 60, 1) 0.0091)**0.0446) 0.0493) Endolyn CD164 CXCR4 1 1.281* 1.158 1.873**** (±0.05, 33,(±0.11, 15, (±0.11, 7, (±0.18, 26, <0.001) 1) 0.0207) 0.0943) TSLP-R —TSLP ND ND ND ND IL33-R T1/ST2 IL33 ND ND ND ND Abbreviations used: SE:Standard Error; ND: Not Detected; CD: Cluster of Differentiation; CT:controls; CCL: C-C motif ligand; CCR: C-C motif ligand receptor; CXCL:C—X—C motif ligand; CXCR: C—X—C motif ligand receptor; CRTH2:Chemoattractant Receptor-homologous molecule expressed on Th2 cells(DP2, PTGDR-2); PGD₂: Prostaglandin D₂; PTGDR: PGD2 receptor; TSLP:Thymic stromal lymphopoietin (-R: receptor); Statistical analyses wereby Mann-Whitney tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P <0.0001.

CXCL12 is described as one of the most overexpressed gene duringperitoneal dialysis and is actively secreted together with PGD₂ duringperitonitis. To study human basophil migration in vivo, we analyzed themboth in blood and in peritoneal dialysis fluid from patients beingtreated for a non-sterile peritonitis. CXCR4 expression was dramaticallyincreased on human basophils recruited to the inflamed peritoneum ascompared to their blood counterparts (data not shown). This basophilrecruitment was associated with a peripheral basopenia (data not shown),as previously shown in active chronic idiopathic urticarial (Jain, S.Dermatology research and practice 2014, 674709), strongly suggestingthat human CXCR4⁺ basophils can migrate in vivo to CXCL12- andPGD₂-secreting inflamed tissues, and that peripheral basopenia reflectsthis active basophil recruitment.

Altogether these data identified both PGD₂/PTGDRs and CXCL12/CXCR4 axesas basophil activation pathways during SLE flares and which may accountfor their associated basopenia. Therefore, these axes may contribute tothe described basophil accumulation in SLOs in individuals with activelupus.

PGD₂/PTGDRs Axis Enhances CXCR4-Dependent Basophil Migration DuringLupus

In order to evaluate the functional consequences of the above findings,ex vivo migration assays of purified basophils from HC and active SLEsubjects were performed. Strikingly, SLE basophils were attracted toCXCL12 gradients while HC basophils did not (FIG. 5a ), reflecting theirdifferences in CXCR4 and CD164 expression (FIG. 3d,f ). However, nodifference was detected with other common basophil chemo-attractantcompounds, including PGD₂ (FIG. 5b ). Since PGD₂ titers were associatedwith basopenia in SLE (FIG. 3c ) and since autoreactive IgE areprevalent in active SLE subjects (FIG. 5c and Dema, B., et al., PLoS One9, e90424 (2014)), we next investigated if these factors couldpotentiate basophil migration towards CXCL12. Standard culture ofpurified human basophils is known to induce intracellular CXCR4externalization, a process inhibited in the presence of IL-3. Primingpurified basophils during 18 hours with 1 μM PGD₂ enhanced their CXCR4expression and their migration towards CXCL12 (FIG. 5d,e ) and inducedPTGDR-2 internalization (data not shown), without inducing theirapoptosis (data not shown). This PGD2 priming induced a slight increasein the high affinity IgE receptor alpha chain (FcεRIα) expression onbasophil surface, which may increase their sensitivity to IgE-dependentstimulation (data not shown). Sub-optimal anti-IgE stimulation(MacGlashan, D., Jr., Clin. Exp. Allergy 40, 1365-1377 (2010)) (i)tended to increase CXCR4 expression on basophils (FIG. 5d ) which mayinfluence their migration towards CXCL12 although statisticalsignificance was not reached (FIG. 5e ), (ii) increased PTGDR-2expression levels on basophils (FIG. 6a ) and (iii) did not inducebasophil degranulation (data not shown). None of other tested compounds(CCL3, CXCL2 and CCL5), known to have an effect on basophils and to bedysregulated during SLE, induced an increased CXCR4 externalization exvivo as PGD2 did (data not shown).

CXCL12 is described as one of the most overexpressed gene duringperitoneal dialysis and is actively secreted together with PGD₂ duringperitonitis. To study human basophils migration in vivo, we analyzedthem both in blood and in peritoneal dialysis fluid from patients beingtreated for a non-sterile peritonitis. CXCR4 was found to bedramatically increased on human basophils recruited to the inflamedperitoneum as compared to their blood counterparts (FIG. 5e ). Thisbasophil recruitment was associated with a peripheral basopenia(supplementary FIG. 5b ), as previously shown in active chronicidiopathic urticaria. This demonstrated that human CXCR4⁺ basophils canmigrate in vivo to CXCL12- and PGD₂-secreting inflamed tissues.

We next studied the mechanism by which PGD₂ induced CXCR4externalization by human basophils. Both PTGDR-1 and PTGDR-2 werecooperatively involved since antagonism of one, the other or bothreceptor(s) led to block CXCR4 externalization (data not shown).Moreover, blocking PTGDRs led to a decreased spontaneous CXCR4externalization. This suggested that either ex vivo culture and/orPGD₂-mediated stimulation of human basophils led them to produce PGD₂ tohave an autocrine effect as eosinophils do. To confirm this hypothesis,we used a specific H-PGDS inhibitor which resulted in the sameinhibition of spontaneous CXCR4 externalization than the one induced bythe PTGDR antagonists, and to a decreased PTGDR-2 internalization (datanot shown). Together with the fact that the H-PGDS inhibitor effect wasovercome only by a ten-fold higher PGD₂ concentration (data not shown),we here confirmed our above hypothesis that PGD₂ led to CXCR4externalization partially by stimulating PGD₂ production by basophilsthemselves.

Altogether, these data strongly suggest that the PGD₂/PTGDRs axisdirectly influences the CXCL12 sensitivity of SLE patient basophils byincreasing both their CXCR4 expression and externalization, and thatlupus environment (including autoreactive IgE, CXCL12 and PGD₂)facilitates this cross-talk resulting in basophil extravasation andperipheral basopenia. Thus, PGD₂ might be required to allowCXCR4-dependent basophil migration to SLOs in SLE individuals. Indeed,both PGD₂/PTGDRs and CXCL12/CXCR4 axes were associated with basopeniaand disease activity in lupus subjects (FIG. 3).

CXCR4/CXCL12 and PGD₂/PTGDRs Axes in Lyn^(−/−) Lupus-Prone Mice

We previously showed that aged Lyn^(−/−) mice develop abasophil-dependent T_(H)2 bias contributing to an IgE-, IL-4- andbasophil-dependent lupus-like nephritis (Charles, N. et al., Nat. Med.16, 701-707 (2010); Charles, N., et al., Immunity 30, 533-543 (2009)).In this lupus-like disease model, basophils accumulate in SLOs leadingto an amplification loop of the disease (Charles, N., et al., Immunity30, 533-543 (2009)).

We next assessed whether this mouse model was involving bothCXCL12/CXCR4 and PGD₂/PTGDRs axes as SLE subjects did. In ex vivomigration assays, Lyn^(−/−) spleen basophils migrated towards CXCL12whereas their WT counterparts did not (FIG. 7a ) mimicking the observeddifferences between SLE subjects and HC (FIG. 5a ). CXCR4 expressionlevels were increased on Lyn^(−/−) basophils from blood, bone marrow(BM) and SLOs as compared to their WT counterparts in aged animals (FIG.7b ). Moreover, CXCR4 expression was increased on WT and Lyn^(−/−)basophils from SLOs as compared to their blood counterparts suggestingan involvement of CXCR4 in their accumulation in these organs (FIG. 7b). CXCL12 intraperitoneal (ip) injection in WT mice induced in vivobasophil migration to the injection site (FIG. 7c ) and to the drainingmesenteric lymph nodes (mLN) (FIG. 8a ).

In vivo i.p. injection of PGD₂ increased CXCR4 expression on mLNLyn^(−/−) basophils (FIG. 7d ), as it did ex vivo on human basophils(FIG. 5d ), and drove their accumulation in SLOs and peritoneum (FIG. 7eand FIG. 8b,c ). Moreover, in vivo ip injection of PGD₂ in Lyn^(−/−)mice led to a significant, but transient, peripheral basopenia whencompared to steady state conditions (data not shown) as observed inperitoneal dialysis patients (data not shown). This PGD₂-inducedbasophil recruitment was strictly dependent on the CXCL12/CXCR4 axissince co-injection with AMD3100, a specific antagonist of CXCR4,completely abolished basophil recruitment both in SLOs and peritoneum(FIG. 7e and FIG. 8b,c ). In mice, PGD₂-induced CXCR4 up-regulation wasas well mediated by both PTGDR-1 and PTGDR-2 (CRTH2), as shown by theeffects of their specific agonists (BW245c and DK-PGD₂, respectively) onspleen WT basophils ex vivo (FIG. 7f ). Both agonists induced asignificant but much lower increase in CXCR4 expression on other WTsplenocytes including T and B cells (data not shown).

PTGDR-1 is known to induce cyclic adenosine monophosphate (cAMP)production upon engagement by PGD₂. We next analyzed the effect of amembrane permeable cAMP (N6,2′-O-dibutyryl-adenosine 3′:5′-cyclicmonophosphate (db-cAMP)) on CXCR4 externalization by mouse WT spleenbasophils ex vivo. Basophils externalized CXCR4 upon db-cAMP exposure(FIG. 9a ) with a 100 fold higher sensitivity than T cells to thiscompound (FIG. 9b ). PGD₂ was unable to induce enough cAMP throughPTGDR-1 to lead to CXCR4 externalization by T cells in these settings,unlike what was observed on basophils (FIG. 9a,b ). Dose responseexperiments of each PTGDR specific agonists in the presence or not ofH-PGDS inhibitor suggested again that both PTGDRs were able tocooperatively induce CXCR4 externalization on basophils throughPTGDR-2-induced PGD₂ synthesis acting in an autocrine way onPTGDR-1-mediated cAMP production (FIG. 9c ).

These results strongly suggest that in Lyn^(−/−) old mice, as in SLEsubjects, PGD₂ enhances in vivo CXCL12-dependent basophil accumulationin inflamed tissues and SLOs by modulating their CXCR4 expression levelsthrough the activation of both PTGDR-1 and PTGDR-2.

PGD₂ Chronic Exposure Accelerates Lupus-Like Disease Development in aBasophil-Dependent Manner

Therefore, a more chronic exposure to PGD₂ in lupus-prone mice beforethey start developing the disease should lead to a chronic accumulationof basophils in SLOs, an increased number of autoreactive plasma cellsand to an acceleration of disease development. To verify thishypothesis, we repeatedly injected ip PGD₂ to young (12 weeks-old)Lyn^(−/−) mice every two days over ten days. As expected, basophilsincreased their CXCR4 expression levels (data not shown) and accumulatedsystemically in SLOs (FIG. 10a,b ) where they were activated as shown bytheir increased IA-IE expression (FIG. 10c ). This was associated withan increased proportion of CD19⁺ CD138⁺ plasma cells in SLOs (FIG. 10d,e), resulting in an increased deposition of immune complexes in thekidney as shown by C3 and IgG deposition quantification (FIG. 10f ).Consequently, nearly all the PGD₂ injected Lyn^(−/−) mice had increasedalbuminuria unlike their PBS-injected counterparts at the end of theprotocol (FIG. 10g ). Other immune cell types analyzed didn't show anysignificant increase in their CXCR4 surface expression (data not shown).Importantly, this PGD₂-induced lupus-like disease acceleration wasdependent on basophils since antibody-mediated (MAR-1) basophildepletion during the whole protocol led to a complete rescue of the PGD₂effects on disease development (FIG. 10b-e ). These results confirmedthat PGD₂, by enabling CXCR4-dependent basophil accumulation in SLOs,contributes to lupus-like disease and to autoantibody-mediated kidneydamage.

Targeting the PGD₂ Axis Reduces CXCR4-Mediated Basophil Accumulation inSLOs and Dampens Lupus-Like Disease

Targeting the CXCL12/CXCR4 axis in murine lupus has already beendescribed and showed some efficacy on disease activity (Balabanian, K.,et al., J. Immunol. 170, 3392-3400 (2003), Wang, A., et al., J. Immunol.182, 4448-4458 (2009)). However, CXCR4 antagonism (with AMD3100),initially developed as an anti-HIV drug, is known to induce a release ofhematopoietic stem cells and interfere with homeostatic functions (Devi,S. et al. J. Exp. Med. 210, 2321-2336, (2013), Hummel, S. et al., Curr.Opin. Hematol. 21, 29-36 (2014)). Preventing the CXCR4 up-regulation onbasophils from lupus-prone Lyn^(−/−) mice by blocking the PGD₂/PTGDRsaxis seemed a safer approach to disable the basophil-dependentamplification loop of autoantibody production in SLOs.

Then, we treated aged Lyn^(−/−) and WT mice by oral gavage with bothspecific antagonists of PTGDR-1 and PTGDR-2, Laropiprant and CAY10471,respectively, at a dose of 5 mg/kg each, twice daily for ten days. Thistreatment led to a dramatic reduction in basophil numbers in SLOs ofLyn^(−/−) animals (FIG. 12a,b ) associated with a decreased CXCR4expression on spleen basophils (FIG. 12c ). Of note, BM and bloodbasophil proportions were not affected by the treatment (FIG. 13a,b ).These results validated the approach consisting of disabling basophilaccumulation in SLOs by targeting the PTGDRs.

As expected, this reduction in basophil accumulation was associated witha significant decrease of CD19⁺CD138⁺ short-lived plasma cell numbers(FIG. 12d and FIG. 13c ). Strikingly, proportions of all other immunecell populations analyzed (B cells, neutrophils, Ly6C⁺ monocytes, andLy6C⁻ monocytes) remained unaffected by the treatment as did their CXCR4expression levels (data not shown). PTGDRs blockade in Lyn^(−/−)animals, by disabling basophil accumulation in SLOs, decreased theirautoantibody titers (FIG. 12e and FIG. 13d ) and their T_(H)2 bias asmeasured by total IgE plasma concentrations (FIG. 12f ). Consequently,this treatment allowed as well a significant decrease in the kidneycontent of C3 and IgG deposits (FIG. 11) and the pro-inflammatorycytokines IL-4 and IL-1β (FIG. 12g,h ). Therefore, a short-termtreatment with PTGDRs antagonists allowed an efficient dampening in thedisease activity observed in our lupus-prone animals.

Altogether, these results suggest that aiming the PGD₂/PTGDRs axis couldbe a valuable new therapeutic approach in SLE. Indeed, PTGDRs blockade,by breaking the CXCL12-dependent basophil homing to SLOs, might turn-offthe basophil-dependent amplification loop of autoantibody production,efficiently preventing flares and subsequent organ damage in SLE (FIG.14).

1. A method for preventing and/or treating SLE in a patient in needthereof, wherein said method comprises administering said patient with aPTGDR-1 antagonist.
 2. The method according to claim 1, wherein saidantagonist is a small molecule antagonist.
 3. The method according toclaim 1, wherein said antagonist has formula (I)

or pharmaceutically acceptable salts thereof, wherein n is 0 or 1; m is1, 2 or 3; R₁ is H, C₁-C₃ alkyl, halogenated C₁-C₃ alkyl or cyclopropyl;R2 is 4-chlorophenyl or 2,4,6-trichlorophenyl.
 4. The method accordingto claim 1, wherein said PTGDR-1 antagonist is laropiprant.
 5. Themethod according to claim 1, wherein said PTGDR-1 antagonist is used incombination with at least a PTGDR-2 antagonist.
 6. The method accordingto claim 5, wherein said PTGDR-2 antagonist has formula formula (VIII)

wherein R1 is H, fluorine, methyl, methoxy, benzyloxy, or hydroxyl, R2is phenyl which is substituted by fluorine, chlorine, trifluoromethyl,methyl, ethyl, propyl, isopropyl, or methoxy, and Y is 0 or 1, orpharmaceutically acceptable salts thereof.
 7. The method according toclaim 6, wherein said PTGDR-2 antagonist is CAY10471 (TM30089).
 8. Themethod according to claim 5, wherein said PTGDR-1 antagonist and PTGDR-2antagonist are formulated in a single pharmaceutical composition.
 9. Themethod according to claim 5, wherein said PTGDR-1 antagonist and PTGDR-2antagonist are formulated in separate pharmaceutical compositions forsimultaneous use, separate use, or use spread over time.
 10. The methodaccording to claim 1, wherein said PTGDR-1 antagonist is a dualPTGDR-1/PTGDR-2 antagonist.
 11. A method for preventing and/or treatingSLE in a patient in need thereof, wherein said method comprisesadministering said patient with a pharmaceutical composition comprisinga PTGDR-1 antagonist and a PTGDR-2 antagonist, or a dual PTGDR-1/PTGDR-2antagonist.
 12. A method for preventing and/or treating SLE in a patientin need thereof, wherein said method comprises administering saidpatient with a PTGDR-1 antagonist and a PTGDR-2 antagonist as a combinedpreparation for simultaneous use, separate use, or use spread over time.13. A method for preventing and/or treating SLE in a patient in needthereof, wherein said method comprises administering said patient with aPTGDR-2 antagonist.
 14. The method according to claim 1, wherein thePTGDR-1 antagonist prevents basophil homing to secondary lymphoidorgans.
 15. The method according to claim 1, wherein the PTGDR-1antagonist prevents, limits the extent or reduces the increase inautoantibody titers and/or the occurrence of SLE flares and/or organdamages.